Marsilea Vestita Research Articles

THE DEVELOPMENT OF THE SPOROCARP OF MARSILEA VESTITA

The tissues of the sporocarp of Marsilea vestita undergo profound changes during development. Early in development, the cells of the peripheral tissues, epidermis, hypodermis and layers of the transitional zone between the hypodermis and more internal tissues contain prominent vacuolar bodies. As development proceeds, these vacuolar bodies disappear. Prominent amyloplasts are found only in the guard cells and in the cells of the transitional zone. Later in development the cells of the hypodermis divide periclinally forming two layers which differentiate as macrosclereids. The cells of the outermost layer of the transitional zone differentiate as osteosclereids. Internally, the cells of the sorophore accumulate large amounts of mucilage in the central vacuoles. The peripheral cytoplasm ultimately degenerates leaving just hygroscopic mucilage. The mucilage carbohydrate contains the sugars, rhamnose and arabinose. In the young sorus, only the spore mother cells and the cells of the indusium contain amyloplasts. By the time of meiosis, there is a massive accumulation of starch in the receptacle, stalk and jacket but not in the tapetum of the sporangia. Late in development, the starch disappears and the mega‐ and microspores become coated with carbohydrate.

The fine structure of fertilization in the fern Marsilea vestita.

The ultrastructural details of fertilization in the fern Marsilea vestita, including gamete approach and fusion, the fate of the spermatozoid organelles and the development of a possible block to polyspermy are described. The spermatozoid approaches the egg through layers of mucilage that surround the megaspores. It moves down the neck of the archegonium into the cavity above the egg. In order to reach the egg, it must move through a small hole in the thick wall that lies across the top of the egg. The fusion of the plasma membranes of the gametes results in an outflow of egg cytoplasm into the clear space under the sperm plasma membrane, creating a fertilization cone. All the organelles of the fertilizing spermatozoid, including nucleus, mitochondrion, microtubule ribbon, multilayered structure, and flagellar band, with approximately 150 flagella, enter the egg cytoplasm. The nucleus enters as a condensed rod of chromatin with no nuclear envelope. The chromatin begins to disperse immediately and a new nuclear envelope is formed around the chromatin by egg endoplasmic reticulum. The mitochondrion and the microtubules of the ribbon and flagella are broken down, but the fates of the flagellar band and the multilayered structure have not been determined. After spermatozoid penetration, a new extracellular layer appears above the surface of the egg, beginning in the region of sperm penetration and spreading across the top of the egg. This layer may be important in preventing other spermatozoids from fusing with the egg.

Analyse expérimentale du developpement in vitro d'embryons isolés de Marsileacées

Summary The culture of Marsilea vestita embryo, in vitro, pointed out the influence of the prothallium cells in the early development. The leaf initiation and the heteroblastic development are influenced by tile intimate and early contact of the shoot apex and of the leaf primordia with the culture medium. Also, shoot apex activity appears to be influenced by modifications of the external environment.

Etude de l’organogenèse radiculaire chez le Marsilea vestita

Histochemical and cytochemieal techniques, together with mapping of mitotic activity and sites of DNA synthesis in embryonic and adult roots of Marsilea vestita help to reveal basic features of their organogenesis. Most of the root comes from its apical cell, but initially its basal portion arises through mitotic activity of the ground tissue of the embryo or stem. Thus the apical cell, although it takes a prominent part in originating root tissues is not the source of the whole of those of the young root. It appears to assume some inductive role, insofar as among the two cells of the root pole of the 8-celled embryo, only that differentiating an apical cell gives rise to a root. In the same way, the apical cell seems to regulate histological differentiation of its surrounding tissues in the elongating root of the adult plant, as well as producing most of these. The apical cell remains active all through the growing phase of the root. Only very late in the development of the latter does it become imperfectly quiescent.

Re-Introduction of Marsilea vestita into Florida

Any area with a poorly documented flora inevitably accumulates in its list of presumed species an assortment of one-time horticultural escapes, ballast waifs, railroad travelers, agricultural relics, and other chance introductions that are incapable of persistence and hence are not properly full-fledged components of area's flora. Such plants, once reported, tend to persist in listings long after they have disappeared in field. Little acclaim is accorded writer who omits a species that he feels is not adequately documented as a present member of his flora, while a degree of pride and even a measure of competitiveness accompanies report of a species in an area where it has not previously been known. Thus it is satisfying to provide first record of Marsilea as a current member of Florida flora since it was reported for state in 1890's. In 1891 L. M. Underwood traveled by rail through Florida, stopping at every opportunity to collect ferns and other vascular plants. In January and in March he paused at a depot known as Orange Bend, in Lake County, central peninsular Florida. Near depot he found in abundance what he identified as the mucronata form of Marsilea vestita. Underwood (1892) published a report of his discovery, noting that his specimens (now at PH, US, YU, and perhaps elsewhere) were first for this species from east of Mississippi River. Guided by Underwood's account, G. V. Nash visited same location on May 16, 1894. He remarked (1895, p. 161): plant occurs along track on both sides of depot for about one-quarter of a mile. It is confined to that limited area so far as I could find out. Its occurrence at such a distance from its ordinary range and its limitation to this small section point very strongly to its being introduced. Nash's specimens (PH, US) bear location Eustis, but are surely from Orange Bend station. A much earlier indication of Marsilea in Florida was published by D. C. Eaton (1872), a quarter century before Underwood's report, but has been wholly overlooked by later authors. Eaton possessed a plant from Florida labeled Marsilea quadrifolia L. (a European species sparingly introduced into northeastern North America), which he thought more likely to be one of western species, M. uncinata Braun, for instance. Two sheets in Eaton herbarium (YU) provide basis for this brief report. Both are Marsilea vestita (and are so annotated by D. S. Correll), and bear printed label: Plantae Floridanae: prope Apalachicola [Franklin County], coll. A. W. Chapman, M.D., 1860. Chapman (1897, p. 640), however, acknowledged Marsilea in southeastern United States only near Vicksburg, Mississippi. The labels on Eaton collections were prepared for his herbarium, and it is possible that an error occurred in documentation of

The blepharoplast of Marsilea: its de novo formation and spindle association.

Blepharoplast formation and function has been investigated by light and electron microscopy in developing microspores of the water fern Marsilea vestita. When single-celled microspores are hydrated they undergo a series of 9 cell divisions producing 7 sterile cells plus 32 spermatids. The blepharoplast, a densely staining, spherical inclusion (0-5-1-0 mum in diameter) interpenetrated by numerous lightly stained channels, is a precursor organelle for the production of the 100-150 basal bodies of the motile sperm. During development it arises twice in the dividing spermatogenous cells; first in telophase of the second to last or 7th division, and again in telophase of the next to last or 8th division. The blepharoplast first forms on the distal side of the nucleus during telophase of the 7th division but soon degenerates as the cell enters prophase and metaphase of the 8th division. The blepharoplast first forms on the distal side of the nucleus during telophase of the 7th division but soon degenerates as the cell enters prophase and metaphase of the 8th division. During telophase of the 8th division the blepharoplast again arises in the cytoplasm near a small indentation of the nuclear envelope. The forming blepharoplast consists of a sphere of lightly stained flocculent material within which 2 plaques separated by 40-50 nm arise. Each plaque possesses 2 dense layers (20 nm) separated by a light layer (10 nm). During subsequent development the distal layer of each plaque becomes progressively more densely stained. Light-staining channels appear within the accumulating dense material a-d 2 hemispherical blepharoplasts emerge. The blepharoplasts become spherical through continued growth and finally separate from each other as they move to the spindle poles during prophase of the last or 9th division. The blepharoplast appears to act as a microtubule organizing centre during formation of the spindle apparatus in prophase of the 9th division. While it remains at the pole throughout mitosis it does not continue to serve as the focal point for the spindle tubules. During metaphase-anaphase of the 9th division the blepharoplast swells, and the channels at its surface begin transforming into procentrioles and finally basal bodies. The results support the contention that basal bodies in Marsilea arise de novo, since no preexisting template such as a centriolar pinwheel is observed and sine the intermediates which initially occur are structurally dissimilar from a procentriole.

Developmental mechanisms in heterospory. III. The plastid cycle during megasporogenesis in Isoetes.

The megasporocyte of Isoetes englemanni at the leptotene-zygotene interval of meiosis contains 4 disk-shaped proplastids about 12 mum in diameter. The disposition of these organelles in the cell is such that each of the four megaspores delimited during cytokinesis contains a single proplastid. During prophase and following their incorporation into the spores, the proplastids are undergoing fission by budding. The buds are first discernible as low surface evaginations which contain a complement of granular somal material, some wefts of tubular membrane and osmiophilic globuli, in addition to a number of vesicles derived by invagination from the inner membrane of the proplastid envelope. As the evaginations emerge they enlarge and the link with the parent body is reduced to a narrow channel. At this stage one or more of the vesicles derived from the proplastid envelope comes into register with the lumen of the channel. One vesicle is transported into the lumen, elongating as it passes through. The passage of the vesicle into the channel destroys the connexion between the matrix of the evagination and the stroma of the proplastid. The occurrence in the cytoplasm around the proplastid of bodies not connected to the proplastid, but identical in structure to the evaginations and carrying a membranous tail suggests that the evaginations are released by abscission of the channel close to the surface of the parent body. After release the bodies undergo division by constriction. Regression of the tail follows division in those bodies which are regular in outline and in which the matrix is ultrastructurally similar to the stroma of the parent organelle. The process does not seem to occur in co-existing forms which have assumed an irregular outline and have a less-opque matrix. The more mature megaspore of Isoetes contains proplastids up to 4 mum in greatest dimension. The stroma in these is dense and granular and contains membrane-bound vesicles, osmiophilic globuli, starch granules and wefts of tubular membrane. There is no evidence that the large budding organelle persists to this later stage in development. The resemblance of the plastids in the more mature megaspore to the bodies produced by evagination earlier in development suggests a common identity. The observations and interpretations lead to the proposition that the plastids in Isoetes englemanni are autonomous. This situation contrasts with the one described for another heterosporous haploid dioecious pteridophyte, Marsilea vestita, where nucleocytoplasmic interaction has been interpreted as the de novo creation of plastids and mitochondria following the elimination by autophagy of the organelles inherited at meiosis. It is suggested that an explanation to account for the 2 different mechanisms might be sought in regard to the degree of developmental success enjoyed by the individual megaspores in the 2 plants. In Isoetes all 4 megaspores of every tetrad survive and develop, while in Marsilea the mature megasporangium contains a single functional megaspore.

An ultrastructural study of the spermatozoid of the fern, Marsilea vestita.

The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.

THE LOCOMOTOR BEHAVIOR OF LYGODIUM AND MARSILEA SPERM

Flagella of living sperm of the ferns, Lygodium japonicum (Thunb.) Sw. and Marsilea vestita Hook, and Grev., beat three dimensionally with a continuous traveling helical wave. The wave is propagated from base to tip of the flagellum. Flagella of Lygodium and Marsilea complete 65 and 30 beat cycles per sec, respectively. Each flagellum circumscribes an open conicoid oriented in a latero‐posterior direction. Only dead sperm have anteriorly directed flagella as illustrated in plant morphology textbooks.

EFFECTS OF CARBON DIOXIDE ON THE GROWTH AND MORPHOGENESIS OF MARSILEA

Increased concentrations of CO2 in air (1–50%) cause young plants of Marsilea vestita to exhibit many characteristics of the water form when they are grown on a solid substrate under sterile conditions. Thus these plants have longer internodes, shorter petioles, more rectangular‐shaped epidermal cells and fewer stomata on the lower leaf epidermis than controls grown in 0.03% CO2. Over a 2‐week period, dry weight increase is considerably greater in 12.5% and 25% CO2 than in 0.03% CO2. Fifty percent CO2 is inhibitory to growth. CO2‐enriched air has the same morphogenetic effect when supplied in the light or the dark. Possible explanations for this effect are discussed.

A CORRELATION BETWEEN THE APICAL CELL AND THE HETEROBLASTIC LEAF SERIES IN MARSILEA

Plants of three species of Marsilea (M. vestita, M. villosa, M. drummondii) were grown in sterile culture under controlled conditions, and stem apices were sampled at one of the three heteroblastic leaf forms typical of this plant: spatulate, bifid, or quadrifid leaves. Statistical analyses were made of the relationship between the area of the apical cell and the leaf form which the plant produces under varied growth conditions. For all three species there is a statistically significant correlation (1% level) between apical cell area and leaf form. The analysis indicates that 83% of the variation in apical cell area in Marsilea vestita, 52% in M. villosa, and 54% in M. drummondii can be related to the change in leaf form. An increase in the glucose concentration of the culture medium increases the average apical cell area, and the addition of the protein synthesis inhibitor 2‐thiouracil at concentrations of 10 mg/liter and 25 mg/liter decreases the average apical cell area. A certain average apical cell area is necessary for the production of a particular leaf form in the series. The area is a relative rather than an absolute size under any one particular growth condition. Any growth condition which inhibits or reverses the increase in apical cell size (which is typical of the normal growth pattern) will inhibit or reverse the normal heteroblastic series. Under constant conditions, the average size at which particular leaf forms develop appears to be species specific. These results confirm a generally held idea that the apex size is related to the heteroblastic leaf series, and they indicate that the area of the apical cell is the key factor rather than the volume or the number of cells of the apex. Undoubtedly the area of the apical cell is only a reflection of the physiological or morphological characteristics of the apical meristem that underlie the heteroblastic leaf series and which currently do not appear to lend themselves to more direct quantitative analysis.

A Correlation between the Apical Cell and the Heteroblastic Leaf Series in Marsilea

Plants of three species of Marsilea (M. vestita, M. villosa, M. drummondii) were grown in sterile culture under controlled conditions, and stem apices were sampled at one of the three heteroblastic leaf forms typical of this plant: spatulate, bifid, or quadrifid leaves. Statistical analyses were made of the relationship between the area of the apical cell and the leaf form which the plant produces under varied growth conditions. For all three species there is a statistically significant correlation (1% level) between apical cell area and leaf form. The analysis indicates that 83% of the variation in apical cell area in Marsilea vestita, 52% in M. villosa, and 54% in M. drummondii can be related to the change in leaf form. An increase in the glucose concentration of the culture medium increases the average apical cell area, and the addition of the protein synthesis inhibitor 2-thiouracil at concentrations of 10 mg/liter and 25 mg/liter decreases the average apical cell area. A certain average apical cell area is necessary for the production of a particular leaf form in the series. The area is a relative rather than an absolute size under any one particular growth condition. Any growth condition which inhibits or reverses the increase in apical cell size (which is typical of the normal growth pattern) will inhibit or reverse the normal heteroblastic series. Under constant conditions, the average size at which particular leaf forms develop appears to be species specific. These results confirm a generally held idea that the apex size is related to the heteroblastic leaf series, and they indicate that the area of the apical cell is the key factor rather than the volume or the number of cells of the apex. Undoubtedly the area of the apical cell is only a reflection of the physiological or morphological characteristics of the apical meristem that underlie the heteroblastic leaf series and which currently do not appear to lend themselves to more direct quantitative analysis.

The Correlation of Physiological Differences and Varions Leaf Forms of an Aqnatic Plant

AbstractAn analysis of the celluar constituents of the leaves of an aquatic fern, Marsilea vestita, was done after and during the formation of different leaf forms.Land leaves of Marsilea produced almost twice as much dry material, soluble carbohydrate, insoluble protein and soluble peptides compared to submerged leaves on a fresh weight basis, but on a dry weight basis there is tittle difference between the two types of leaves. the submerged leaf soluble nitrogen fraction consisted mostly of ammonia and free amino acids, while that of the land leaf probably con tained mostly peptides. Free arginine was present in large quantities in megaspores and as the plant matured, at the expense of the megaspore, the amount of arginine left in the pool diminished. A correlation was found between soluble sugar increase and leaf elongation, Just prior to the production of submerged leaves the developing leaves contained low levels of protein and soluble sugar, but high levels of soluble nitrogen and an increased amount of starch. A theory which involves metabolism was proposed to account for the leaf form changes in Marsilea.

OBSERVATIONS ON THE MORPHOLOGY AND PHYSIOLOGY OF MARSILEA SPERM

The multiciliated sperm of the water fern Marsilea vestita was examined with a view to establishing its suitability as an experimental subject. Time‐course experiments revealed spermatid development to be temperature dependent. Sterile techniques were devised for observation of sperm on both a population and an individual basis. Sperm discharge, active and senescing sperm were examined by phase‐contrast microscopy. A regular pattern of senescence was ascertained. This included vacuolation of the cytoplasmic vesicle, loss of motility, and ultimate loss of the helical structure of the sperm coil. Sperm life spans were recorded using motility and O2 uptake as criteria. Sperm populations are active 3–3½ hr at ambient temperature (22–25 C). Individual sperm are active less than 1 hr. Sperm suspensions show a decline in O2 uptake which parallels the loss of motility. Various constituents affecting the life span were investigated. A twofold prolongation of the sperm life span occurred in the presence of 0.1 m sucrose. An ultrastructural examination of the mature sperm was made to aid in assessing its metabolic potential. The sperm shows little ultrastructural differentiation. The cytoplasmic vesicle is predominantly composed of starch‐containing plastids. The main structural components of the sperm coil are a continuous mitochondrial band, an elongate nucleus, and a series of microtubules which separate the basal bodies from the nucleus and mitochondrion. A comparison of ultrastructural features common to Pteridium and Marsilea was made and factors affecting senescence discussed.

Observations on the Morphology and Physiology of Marsilea Sperm

The multiciliated sperm of the water fern Marsilea vestita was examined with a view to establishing its suitability as an experimental subject. Time-course experiments revealed spermatid development to be temperature dependent. Sterile techniques were devised for observation of sperm on both a population and an individual basis. Sperm discharge, active and senescing sperm were examined by phase-contrast microscopy. A regular pattern of senescence was ascertained. This included vacuolation of the cytoplasmic vesicle, loss of motility, and ultimate loss of the helical structure of the sperm coil. Sperm life spans were recorded using motility and O2 uptake as criteria. Sperm populations are active 3–3½ hr at ambient temperature (22–25 C). Individual sperm are active less than 1 hr. Sperm suspensions show a decline in O2 uptake which parallels the loss of motility. Various constituents affecting the life span were investigated. A twofold prolongation of the sperm life span occurred in the presence of 0.1 m sucrose. An ultrastructural examination of the mature sperm was made to aid in assessing its metabolic potential. The sperm shows little ultrastructural differentiation. The cytoplasmic vesicle is predominantly composed of starch-containing plastids. The main structural components of the sperm coil are a continuous mitochondrial band, an elongate nucleus, and a series of microtubules which separate the basal bodies from the nucleus and mitochondrion. A comparison of ultrastructural features common to Pteridium and Marsilea was made and factors affecting senescence discussed.

THE GROWTH OF EXCISED LEAVES OF MARSILEA

Excised young submerged leaves of Marsilea vestita grew to sizes comparable to that reached on intact plants and lived for approximately 20 days if supplied with auxin and coconut water in a liquid medium.

Changes in the Shoot Apex during the Early Development of the Fern Marsilea vestita

Marsilea is an aquatic fern which can be grown in sterile culture and which passes through a juvenile phase before reaching adulthood. It is therefore an ideal plant to use in examining the events at the shoot apex which lead up to the production of juvenile and, later, adult leaves. Allsopp1 has suggested that in Marsilea a small apical meristem initiates primordia with a relatively limited capacity for development, and Cutter2 states in her review: “It is thus held that there is an important correlation between the size of the shoot apex and the extent of development of the leaf primordia to which it gives rise, leaf primordia formed on a large apex being capable of developing into adult leaves, whereas those formed on a small apex are considered to have only a limited capacity for growth and development. Apical regions of well-nourished sporelings with adult leaves are seen by inspection to be larger than those of less-well-developed sporelings with juvenile leaves, but no detailed study of changes in size of the apex in Marsilea has yet been undertaken”. The object of this communication is to find out whether there is any correlation between the size of the shoot apex and the production of juvenile or adult leaves.

THE MORPHOLOGICAL EFFECTS OF PROTEIN SYNTHESIS INHIBITION IN MARSILEA

Marsilea vestita and M. drummondii were grown in sterile cultures to which concentrations of the protein synthesis inhibitors, 2‐thiouracil (10 mg/liter) and 5‐fluorouracil (1 mg/liter) had been added. When young sporelings are grown in a solution of thiouracil at optimum concentration, there is an inhibition of the rate of leaf formation, a retardation of the leaf heteroblastic series, and all leaves develop as land forms. When thiouracil is added to plants which are already producing typical adult, quadrifid leaves, the effects depend on whether the treated plants are water or land forms. Plants which are typically water forms convert to land forms. After treatment successive leaves develop typical sunken stomata on both leaf surfaces. The tissues of the rhizome, root and petiole are more compact and, in general, the cells of the plant have thicker walls. Vascular patterns are not changed, though the size of the rhizome, root and petiole may be reduced. Plants which are typically land forms are less affected than the water forms, but they show a small reduction in apex volume and an apparent reversion of the leaflet number from the typical quadrifid leaf to a trifid, bifid, or single lamina condition. In both land and water forms apical dominance may be broken by treatment with 10 mg/liter thiouracil or 1 mg/liter fluorouracil and numerous lateral branches develop. Higher concentrations (15–25 mg/liter of thiouracil) may result in abnormal development of lateral axillary buds, petiole bases and leaflets. The meristems of the plant are differentially sensitive to thiouracil; leaflet meristems are most sensitive, the root meristems are the least sensitive. It appears that a true reversion to juvenile leaf development need not occur even though protein synthesis and the volume of the apex are reduced. The development of the land or water form in Marsilea appears to depend on rate of growth. Hence inhibition of the growth of typical water forms, through inhibition of protein synthesis, causes a shift in development toward the morphology typical of land forms.

MORPHOLOGY OF MARSILEA VESTITA. III. MORPHOGENESIS OF THE LEAVES OF ETIOLATED PLANTS

The laminae of etiolated Marsilea vestita leaves develop by means of marginal meristems. Unlike light‐grown plants, the form of the etiolated plant is not affected by growth on a solid medium. All of the young leaves isolated from light‐grown submerged plants will elongate in darkness. The smallest, etiolated, uncoiled leaves develop into land leaves when they are placed in light, and this development occurs regardless of whether the leaves remain on the plant or are isolated on nutrient agar. Only these smallest leaves (2.3 mm average length) are actually capable of being converted from a submerged leaf form to a land leaf form by darkness.

The Effect of Various Environmental Factors on the Leaf Form of the Aquatic Fern Marsilea vestita

The Effect of Various Environmental Factors on the Leaf Form of the Aquatic Fern <i>Marsilea vestita</i>